Cervical Tissue Derived Organ Culture to Test Microbicides

宫颈组织衍生器官培养以测试杀菌剂

• 批准号: 7681868
• 负责人: Phalguni GUPTA
• 金额: $ 29.26万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7681868
• 关键词: Antiviral Agents; Applications Grants; Bacteria; Biological Assay; Cell Culture System; Cell Culture Techniques; Cells; Cervical; Cervix Uteri; Clinical Trials; Collaborations; Complement; Data; Development; Drug Formulations; Environmental Risk Factor; Epithelium; Evaluation; Genital system; HIV; HIV-1; HIV-1 vaccine; Histocompatibility; Histology; IL8 gene; Immune; In Vitro; Infection; Inflammation; Inflammatory; Inflammatory Response Pathway; Interleukin-6; Measures; Memory; Modeling; Monitor; Monkeys; Mucous Membrane; Neisseria gonorrhoeae; Nucleosides; Organ Culture Techniques; Outcome; Outcome Study; Peptides; Physiological; Prevotella melaninogenica; Principal Investigator; Property; Reverse Transcriptase Inhibitors; Screening procedure; Seminal fluid; Sexually Transmitted Diseases; System; Testing; Time; Tissues; Topical application; Toxic effect; Vagina; Vaginal Ring; anti-HIV microbicide; antimicrobial peptide; base; cell injury; cytokine; cytotoxic; cytotoxicity; cytotoxicity test; genital secretion; in vitro Assay; in vivo; inhibitor/antagonist; microbicide; microorganism; non-nucleoside reverse transcriptase inhibitors; pinacolyl methylphosphonic acid; preclinical evaluation; prevent; programs; response; retrocyclin; simian human immunodeficiency virus; tool; transmission process; vaginal fluid; vaginal microbicide

The in vitro cell culture system has been widely used as a primary screening tool for evaluating anti-HIV-1 activity of microbicides. However, there is clearly a need to develop a vaginal and cervical tissue based in vitro system to test the cytotoxicity and antiviral activity in the context of the complete tissue matrix. We have recently developed a cervical tissue-derived organ culture model which mimics in vivo conditions and has been used to test microbicides for their ability to block HIV-1 transmission and measure inflammatory cytokines in response to microbicides and sexually transmitted infection-related bacteria. Our hypothesis is that a cervical tissue-based organ culture is an ideal system to test microbicides for toxicity in genital tissue and for its ability to block transmission of HIV-1 with varying phenotypic properties across the cervical epithelium in the presence of common environmental factors that are present in vagina, such as semen, vaginal fluid, and STI-related microorganisms. Specific aims of the project are: 1) Evaluation of -nucleoside reverse transcriptase inhibitor (NNRTI) 5-chloro-3-phenylsulfonylindole-2-carboxamide (CSIC) from Project 1 and the entry inhibitor antimicrobial peptide retrocyclin RC101 from Project 2 alone or in combination and their formulations for their ability to block HIV-1 transmission across the mucosa in a cervical tissue-based organ culture. 2) Assessment of cervical tissue inflammation and their changes in response to CSIC and RC101 from Projects 1 and 2, respectively, using the organ culture model. The expression of proinflammatory cytokines, such as 11-13, IL-6, IL-8 and TNF-a will be monitored by measuring their messages in the tissues by the real time PCR and secretion in the culture supernatant using the Luminex system; 3) Evaluation of anti-HIV activity of CSIC and RC101 under physiologically relevant conditions. CSIC and RC101 alone or in combination and their formulations will be evaluated for their antiviral activities in the presence of semen and vaginal fluid. In addition the organ culture model will be expanded to measure STIrelated microorganisms, such as Neisseria gonorrhoeae and Prevotella melaninogenica by measuring proinflammatory cytokine response. Microbicides will then be evaluated for their antiviral activity in the aresence of the proinflammatory cytokines induced by these microorganisms.The proposed studies in this Droject will complement various other projects in this U19 grant application by providing a valuable in vitro cervical tissue-based assay which will bridge between the microbicide development (Projects 1 and 2), monkey model (Project 4) and Formulation Core (Core B).

体外细胞培养系统已被广泛用作评估抗HIV-1的初级筛选工具 杀微生物剂的活性。然而，显然需要开发一种基于 体外系统测试完整组织基质中的细胞毒性和抗病毒活性。我们有 最近开发了一种模拟体内条件的宫颈组织衍生器官培养模型， 用于测试杀菌剂阻止 HIV-1 传播和测量炎症的能力 细胞因子对杀微生物剂和性传播感染相关细菌的反应。我们的假设是 基于宫颈组织的器官培养是测试杀菌剂对生殖器组织毒性的理想系统 以及它能够阻止具有不同表型特性的 HIV-1 通过宫颈传播 阴道内存在常见环境因素（例如精液）的情况下，上皮细胞 阴道分泌物和性病相关微生物。该项目的具体目标是： 1) β-核苷的评估 逆转录酶抑制剂 (NNRTI) 5-氯-3-苯磺酰基吲哚-2-甲酰胺 (CSIC)，来自项目 1 以及来自项目2的进入抑制剂抗菌肽逆环素RC101单独或组合，以及 他们的配方能够阻止 HIV-1 在基于宫颈组织的粘膜上传播 器官培养。 2）评估宫颈组织炎症及其对CSIC和的反应​​的变化 RC101 分别来自项目 1 和 2，使用器官培养模型。的表达式为 促炎性细胞因子，如 11-13、IL-6、IL-8 和 TNF-a 将通过测量其浓度来监测 使用 Luminex 通过实时 PCR 检测组织中的信息并在培养物上清液中进行分泌 系统; 3)在生理相关条件下评估CSIC和RC101的抗HIV活性。中船重工 和 RC101 单独或组合，其制剂的抗病毒活性将在 精液和阴道液体的存在。此外，器官培养模型将扩展到测量 STI 相关的 通过测量微生物，例如淋病奈瑟氏菌和产黑色素普雷沃氏菌 促炎细胞因子反应。然后将评估杀菌剂的抗病毒活性 这些微生物诱导促炎细胞因子的出现。本研究中提出的研究 Droject 将通过提供有价值的体外实验来补充 U19 拨款申请中的各种其他项目 基于宫颈组织的检测将在杀菌剂开发之间架起桥梁（项目 1 和 2）， 猴子模型（项目 4）和配方核心（核心 B）。